The prevalence of the Burkholderia cepacia complex (Bcc), a group of multidrug-resistant (MDR) pathogens, is predicted to significantly increase in patients with pulmonary disorders (e.g., chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and asthma) by 2024. Moreover, MDR Bcc isolates that are resistant to all currently recommended therapies are emerging. Unfortunately, the development of novel drugs against MDR Bcc is lacking as is our understanding of these unique pathogens. In a retrospective study, a 35% mortality rate in Veterans that acquired a Bcc infection was observed. Additionally, Veterans are shown to be disproportionately affected by COPD, which puts them at an increased risk of acquiring infections by Bcc. Indeed, the number of Bcc outbreaks around the world has doubled over the last decade. Identifying novel strategies to overcome antibiotic resistance in these highly complex organisms that possess multiple chromosomes is a significant unmet medical need and a substantial scientific challenge. ?-Lactams are one of the most prescribed and safest class of antibiotics and are often used to treat Bcc infections. However, the production of ?-lactamases is the most prevalent ?-lactam-resistance mechanism in members of the Bcc, which possess two chromosomally-encoded inducible ?-lactamases, blapenA and blaampC. As a result, the main objective of this application is to identify novel ways of overcoming ?-lactam resistance in Bcc. Building upon studies performed previously, mechanism-based approaches will be used to selectively inhibit the following proteins in Bcc: 1. PenA, a versatile carbapenemase; 2. AmpC, a unique cephalosporinase; 3. Penicillin binding proteins (PBPs), the biological target of ?-lactams and whose inhibition is linked to bla (?- lactamase gene) expression; and 4. PenRA, the transcription regulator of bla genes. To address these objectives, a mechanism-based approach will be used to restore susceptibility to MDR Bcc by testing selected ?-lactams alone and in combination with ?-lactamase inhibitors, performing biochemical and structural analysis of PenA and AmpC with the ?-lactams and ?-lactamase inhibitors, analyzing the genomes of MDR Bcc, and determining the in vivo efficacy of selected combinations. Moreover, the link between PBP inhibition and bla expression will be deciphered by identifying which ?-lactams effect bla expression, measuring the binding of ?-lactams to PBPs, visualizing cells exposed to ?-lactams via microscopy to reveal the impact of ?-lactams on cell morphology, and constructing pbp gene knockouts and assessing their phenotypes. In addition, PenRA will be targeted for inhibition in B. multivorans by using crystallography to define the binding pocket of the PenRA effector binding domain (EBD) and conducting a targeted small molecule inhibitor library screen using an in-house high-throughput fluorescence assay. The anticipated outcomes include identifying novel combinations to inhibit highly drug resistant Bcc by determining which compounds target PenA, AmpC, and/or PBPs. Moreover, a greater understanding of the link between PBP inhibition and bla expression will be gained, thus allowing clinicians to make better choices for therapy. The interactions between native ligand of PenRA as well as a selected panel of small molecules which resemble the native ligand will be determined, thus allowing for the identification of ?lead? compounds to target PenRA and inhibit bla expression. Based on the studies conducted herein, Veterans as well as other individuals that acquire a Bcc infection will have alternative therapeutic options compared to what is currently available, enabling clinicians to eradicate the organism and obtain clinical cure.